Gene transfer for the correction of inborn errors of metabolism and neurodegenerative diseases of the central nervous system (CNS), and for the treatment of cancer has been accomplished with recombinant adenoviral vectors. High particle doses, however, are required for efficacy in mice and rats, and for the infection of large numbers of cells in monkeys. The delivery of such high particle loads has the negative side effect of inducing an immune response in vivo. Thus, gene transfer to brain tissues with adenovirus type 2 (Ad2) or Ad5 vectors is inefficient, which is also true for endothelia, smooth muscle, and differentiated airway epithelia. Methods that improve the efficiency of adenovirus-mediated gene transfer to cells of the CNS, or other target cells such as tumor cells, could reduce the particle load required to achieve sufficient levels of transduction. Improved efficiency could then reduce toxicity and increase the therapeutic index.
There is a continuing need for vehicles and methods for efficient adenovirus-mediated gene transfer of nucleic acids or proteins to cells, such as cells of the CNS or tumor cells.
The present invention provides adenovirus serotype 30 (Ad30) fiber proteins, such as the polypeptide encoded by SEQ ID NO:1. The present invention also provides a polynucleotide encoding such Ad30 fiber protein, such as the polynucleotide encoded by SEQ ID NO:12. As used herein, the term xe2x80x9cfiber proteinxe2x80x9d includes variants or biologically active or inactive fragments of this polypeptide. A xe2x80x9cvariantxe2x80x9d of the polypeptide is a fiber protein that is not completely identical to a native fiber protein. A variant fiber protein can be obtained by altering the amino acid sequence by insertion, deletion or substitution of one or more amino acid. The amino acid sequence of the protein is modified, for example by substitution, to create a polypeptide having substantially the same or improved qualities as compared to the native polypeptide. The substitution may be a conserved substitution. A xe2x80x9cconserved substitutionxe2x80x9d is a substitution of an amino acid with another amino acid having a similar side chain. A conserved substitution would be a substitution with an amino acid that makes the smallest change possible in the charge of the amino acid or size of the side chain of the amino acid (alternatively, in the size, charge or kind of chemical group within the side chain) such that the overall peptide retains its spacial conformation but has altered biological activity. For example, common conserved changes might be Asp to Glu, Asn or Gln; His to Lys, Arg or Phe; Asn to Gln, Asp or Glu and Ser to Cys, Thr or Gly. Alanine is commonly used to substitute for other amino acids. The 20 essential amino acids can be grouped as follows: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan and methionine having nonpolar side chains; glycine, serine, threonine, cystine, tyrosine, asparagine and glutamine having uncharged polar side chains; aspartate and glutamate having acidic side chains; and lysine, arginine, and histidine having basic side chains. Stryer, L. Biochemistry (2d edition) W. H. Freeman and Co. San Francisco (1981), p. 14-15; Lehninger, A. Biochemistry (2d ed., 1975), p. 73-75.
It is known that variant polypeptides can be obtained based on substituting certain amino acids for other amino acids in the polypeptide structure in order to modify or improve biological activity. For example, through substitution of alternative amino acids, small conformational changes may be conferred upon a polypeptide that result in increased bioactivity. Alternatively, amino acid substitutions in certain polypeptides may be used to provide residues that may then be linked to other molecules to provide peptide-molecule conjugates that retain sufficient properties of the starting polypeptide to be useful for other purposes.
One can use the hydropathic index of amino acids in conferring interactive biological function on a polypeptide, wherein it is found that certain amino acids may be substituted for other amino acids having similar hydropathic indices and still retain a similar biological activity. Alternatively, substitution of like amino acids may be made on the basis of hydrophilicity, particularly where the biological function desired in the polypeptide to be generated in intended for use in immunological embodiments. The greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity. U.S. Pat. No. 4,554,101. Accordingly, it is noted that substitutions can be made based on the hydrophilicity assigned to each amino acid. In using either the hydrophilicity index or hydropathic index, which assigns values to each amino acid, it is preferred to conduct substitutions of amino acids where these values are xc2x12, with xc2x11 being particularly preferred, and those with in xc2x10.5 being the most preferred substitutions.
The variant amino acid molecule of the present invention has at least 50%, at least about 80%, or even at least about 90% but less than 100%, contiguous amino acid sequence homology or identity to the amino acid sequence of a corresponding native nucleic acid molecule or polypeptide.
The amino acid sequence of the variant fiber protein corresponds essentially to the native fiber protein""s amino acid sequence. As used herein xe2x80x9ccorresponds essentially toxe2x80x9d refers to a polypeptide sequence that will elicit a biological response substantially the same as the response generated by native fiber protein. Such a response may be at least 60% of the level generated by native fiber protein, and may even be at least 80% of the level generated by native fiber protein.
A variant of the invention may include amino acid residues not present in the corresponding native fiber protein, or may include deletions relative to the corresponding native fiber protein. A variant may also be a truncated xe2x80x9cfragmentxe2x80x9d as compared to the corresponding native fiber protein, i.e., only a portion of a full-length protein. For, example, the polypeptide of the present invention may contain one or more of the three regions of an Ad30 fiber, i.e., a tail region (such as amino acids 1-45 of SEQ ID NO:1), a shaft region (such as amino acids 46-188 of SEQ ID NO:1) or a knob region (such as amino acids 189-371 of SEQ ID NO:1). Fiber protein variants also include peptides having at least one D-amino acid.
The variant fiber protein of the present invention may be expressed from an isolated DNA sequence encoding the variant fiber protein. The amino acid changes from the native to the variant fiber protein are achieved by changing the codons of the corresponding nucleic acid sequence. xe2x80x9cRecombinantxe2x80x9d is defined as a peptide or nucleic acid produced by the processes of genetic engineering. It should be noted that it is well-known in the art that, due to the redundancy in the genetic code, individual nucleotides can be readily exchanged in a codon, and still result in an identical amino acid sequence. The terms xe2x80x9cprotein,xe2x80x9d xe2x80x9cpeptidexe2x80x9d and xe2x80x9cpolypeptidexe2x80x9d are used interchangeably herein.
The Ad30 fiber protein as described above may be operably linked to an amino acid sequence for a therapeutic agent. An amino acid or nucleic acid is xe2x80x9coperably linkedxe2x80x9d when it is placed into a functional relationship with another amino acid or nucleic acid sequence. For example, DNA a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, xe2x80x9coperably linkedxe2x80x9d means that the amino acid or nucleic acid sequences being linked are contiguous, and, in the case of a secretory leader in DNA, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
As used herein, the term xe2x80x9ctherapeutic agentxe2x80x9d refers to any agent or material that has a beneficial effect on the mammalian recipient. Thus, xe2x80x9ctherapeutic agentxe2x80x9d embraces both therapeutic and prophylactic molecules having nucleic acid or protein components. The mammalian recipient may have a condition that is amenable to gene replacement therapy. As used herein, xe2x80x9cgene replacement therapyxe2x80x9d refers to administration to the recipient of exogenous genetic material encoding a therapeutic agent and subsequent expression of the administered genetic material in situ. Thus, the phrase xe2x80x9ccondition amenable to gene replacement therapyxe2x80x9d embraces conditions such as genetic diseases (i.e., a disease condition that is attributable to one or more gene defects), acquired pathologies (i.e., a pathological condition that is not attributable to an inborn defect), cancers and prophylactic processes (i.e., prevention of a disease or of an undesired medical condition).
According to one embodiment, the mammalian recipient has a genetic disease and the exogenous genetic material comprises a heterologous gene encoding a therapeutic agent for treating the disease. In yet another embodiment, the mammalian recipient has an acquired pathology and the exogenous genetic material comprises a heterologous gene encoding a therapeutic agent for treating the pathology. According to another embodiment, the patient has a cancer and the exogenous genetic material comprises a heterologous gene encoding an anti-neoplastic agent. In yet another embodiment the patient has an undesired medical condition and the exogenous genetic material comprises a heterologous gene encoding a therapeutic agent for treating the condition.
The present invention also provides expression vectors containing an Ad backbone nucleic acid sequence and polynucleotide encoding a chimeric Ad fiber polypeptide comprising a tail region, a shaft region and a knob region, wherein at least one of these regions comprises an Ad30 tail region, an Ad30 shaft region or an Ad30 knob region. The expression vector may also contain a nucleotide sequence encoding a therapeutic agent.
The present invention also provides viral particles and mammalian cells containing the expression vector described above. The cell may be human, and may be from prostate, brain, breast, lung, spleen, kidney, heart, or liver. Alternatively, the cell may be a neuroprgenitor or stem cell.
The present invention also provides a method of transducing cells lacking CAR comprising contacting the cells with an expression vector or virus particle containing Ad backbone nucleic acid sequence and polynucleotide encoding a chimeric Ad fiber polypeptide comprising a tail region, a shaft region and a knob region, wherein at least one of these regions comprises an Ad30 tail region, an Ad30 shaft region or an Ad30 knob region. The cell may be a neuronal or epithelial cell, such as a human umbilical vein epithelial cell (HUVEC), or may be a tumor cell.
The present invention further provides a method of treating a genetic disease or cancer in a mammal by administering a polynucleotide, polypeptide, expression vector, or cell described above. The genetic disease or cancer may be one of the diseases listed in Tables 1-3 below.
In general, the invention relates to polypeptides that can be used as a therapeutic agent, and polynucleotides, expression vectors, virus particles and genetically engineered cells, and the use of them, for expressing the therapeutic agent. In particular, the invention may be used as a method for gene therapy that is capable of both localized and systemic delivery of a therapeutically effective dose of the therapeutic agent.
According to one aspect of the invention, a cell expression system for expressing a therapeutic agent in a mammalian recipient is provided. The expression system (also referred to herein as a xe2x80x9cgenetically modified cellxe2x80x9d) comprises a cell and an expression vector for expressing the therapeutic agent. The expression vector further includes a promoter for controlling transcription of the heterologous gene. The promoter may be an inducible promoter. The expression system is suitable for administration to the mammalian recipient. The expression system may comprises a plurality of non-immortalized genetically modified cells, each cell containing at least one recombinant gene encoding at least one therapeutic agent.
The cell expression system can be formed ex vivo or in vivo. To form the expression system ex vivo, one or more isolated cells are transduced with a virus or transfected with a nucleic acid or plasmid in vitro. The transduced or transfected cells are thereafter expanded in culture and thereafter administered to the mammalian recipient for delivery of the therapeutic agent in situ. The genetically modified cell may be an autologous cell, i.e., the cell is isolated from the mammalian recipient. The genetically modified cell(s) are administered to the recipient by, for example, implanting the cell(s) or a graft (or capsule) including a plurality of the cells into a cell-compatible site of the recipient.
According to yet another aspect of the invention, a method for treating a mammalian recipient in vivo is provided. The method includes introducing an expression vector for expressing a heterologous gene product into a cell of the patient in situ. To form the expression system in vivo, an expression vector for expressing the therapeutic agent is introduced in vivo into target location of the mammalian recipient by, for example, intraperitoneal injection.
The expression vector for expressing the heterologous gene may include an inducible promoter for controlling transcription of the heterologous gene product. Accordingly, delivery of the therapeutic agent in situ is controlled by exposing the cell in situ to conditions that induce transcription of the heterologous gene.
According to yet another embodiment, a pharmaceutical composition is disclosed. The pharmaceutical composition comprises a plurality of the above-described genetically modified cells or polypeptides and a pharmaceutically acceptable carrier. The pharmaceutical composition may be for treating a condition amenable to gene replacement therapy and the exogenous genetic material comprises a heterologous gene encoding a therapeutic agent for treating the condition. The pharmaceutical composition may contain an amount of genetically modified cells or polypeptides sufficient to deliver a therapeutically effective dose of the therapeutic agent to the patient. Exemplary conditions amenable to gene replacement therapy are described below.
According to another aspect of the invention, a method for forming the above-described pharmaceutical composition is provided. The method includes introducing an expression vector for expressing a heterologous gene product into a cell to form a genetically modified cell and placing the genetically modified cell in a pharmaceutically acceptable carrier.
According to still another aspect of the invention, a cell graft is disclosed. The graft comprises a plurality of genetically modified cells attached to a support that is suitable for implantation into the mammalian recipient. The support may be formed of a natural or synthetic material.
According to still another aspect of the invention, an encapsulated cell expression system is disclosed. The encapsulated expression system comprises a plurality of genetically modified cells contained within a capsule that is suitable for implantation into the mammalian recipient. The capsule may be formed of a natural or synthetic material.
These and other aspects of the invention as well as various advantages and utilities will be more apparent with reference to the detailed description of the invention and to the accompanying drawings.